Background: Medication adherence after percutaneous coronary intervention (PCI) is essential to preventing the risk of restenosis. Even though Type D personality and depression have been known to affect medication non-adherence, their combined influence on PCI patients remains unclear. Aim: We aimed to identify how both Type D personality and depression were associated with medication non-adherence for 3 months after successful PCI. Methods: This prospective cohort study included 257 PCI patients, who took 3 or more cardiac medications, at a university hospital. We measured sociodemographic and clinical variables, Type D personality, depression, and medication non-adherence using face-to-face interviews and medical record reviews. Results: The total prevalence of medication non-adherence at the one- and three-month follow-ups was 14% and 16%, respectively. At one month, the prevalence of those with a combination of Type D personality and depression (23.4%) and depression alone (24%) was significantly higher than other groups. At three months, the prevalence of the Type D personality-only group (39.1%) was the highest. Type D personality increased the risk of medication non-adherence 5.089 times at three months, while depression increased it 2.6 times at one month. However, the risk of medication non-adherence was not increased in patients with combined Type D personality and depression. Conclusions: Individual assessments of Type D personality and depression are required. Therefore, psychological interventions focusing on personality and depression are crucial. Longitudinal follow-up studies must explore the interaction or individual impact of Type D personality and depression on medication non-adherence and other negative outcomes. Percutaneous coronary intervention (PCI), using a balloon and stent in the diseased area of the artery, is effective in relieving symptoms such as chest pain, shortness of breath, and nausea . By treaMedication adherence is a particularly important component of self-care after PCI for effective secondary prevention . In addiNon-adherence to medications remains a major problem for cardiovascular patients. It leads to poor clinical outcomes, including rehospitalization, subsequent myocardial infarction (MI), and increased mortality in various patient settings . To imprType D personality, a type personality, is combined by social inhibition  and negative affectivity  . The TypThe prevalence of depression as a mood status in patients with CAD ranges from 25–50% . DepressDespite evidence of these factors’ importance in the outcomes of CAD patients, the prospective relationship between medication non-adherence and Type D personality or/and depression has rarely been reported . MoreoveTo date, even though CAD has become a long term chronic condition, little is known for combined Type D personality and depression in patients after PCI ,27. TherThis was a secondary analysis of data from a prospective cohort study demonstrating that left ventricular ejection fraction and post-cardiac depressive symptoms have a combined effect on major adverse cardiac events after successful primary PCI during a 12-month follow-up in Korea . We usedβ-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, nitrates, calcium channel blockers, antiarrhythmics, diuretics), aged 18 years or above, fluent in Korean, and able to provide informed consent. Patients were excluded from the study if they had cognitive impairment, known alcohol or illicit drug use, or a physical or psychological disability inhibiting communication. At baseline, patients completed measures of Type D personality and depression and provided demographic characteristics. At 1 and 3 months, patients completed a self-reported measure of medication adherence via a face-to-face interview at the outpatient clinic.Inclusion criteria included being on three or more cardiac medications  developed and translated Korean language version by Morisky, Ang, Krousel-Wood, and Ward ,31,32. Tα for the MMAS-8 was 0.83. In this study, Cronbach’s α was 0.58.It is a self-report questionnaire with 8 questions (items). Medication non-adherence is detected by an MMAS-8 score <6 and medication adherence is detected by an MMAS-8 score ≥6. Cronbach’s α for the scale was 0.95.For Type D personality, the DS-14 developed by Denollet  was usedα for the scale was 0.84.The Patient Health Questionnaire (PHQ-9) is a 9-item self-reported questionnaire designed to evaluate the presence of depressive symptoms over the prior two weeks . In thisp values < 0.05 were considered statistically significant. Categorical data were presented as percentages and continuous data as mean (SD). Differences between continuous variables were assessed using a t-test, and the x2 test (or Fisher’s exact test when appropriate) was used for categorical variables.All analyses were undertaken using SPSS Version 23 . To measure medication adherence, we used a repeated measures analysis of variance (ANOVA) and a one-way ANOVA performed by combined Type D personality and depression groups. Univariate and multivariate logistic regression analysis was conducted with medication adherence at 1 and 3 months as the dependent variable. We adjusted for age, sex , educational level , exercise , and perceived health status .The investigation conforms to the principles outlined in the Declaration of Helsinki. Ethical approval for secondary analysis was granted by the Institutional Review Board of Gachon University in May 2018 (IRB No.1044396-201804-HR-087-01). Patients were given an information sheet about the study by cardiology staff who would normally be involved in their care. If they wished to participate, a meeting was set up with a researcher where further information about the study was provided and written informed consent was obtained.p = 0.044). The lowest educational level was observed in the combined Type D personality and depression group (p = 0.001). At 24%, the proportion of patients who exercised (p = 0.010) was the lowest in the Type D personality and depression group.A total of 257 patients who underwent PCI were included in this study . The totp = 0.010).Among clinical variables, 80.5% of the participants were diagnosed with unstable angina and 19.5% with acute MI. The group with combined Type D personality and depression showed the lowest percentage of perceived good health status (p = 0.049). The Type D personality-only group displayed the highest (39.1%) medication non-adherence at the 3-month follow-up (p = 0.013).We classified medication non-adherence as a score of <6; the frequency by baseline Type D personality and depression at 1- and 3-month follow-ups is demonstrated in p = 0.004). Medication adherence demonstrated statistically significant differences among the 4 groups (p = 0.025). Especially, at a mean of 6.49, medication adherence was significantly lower in the depression group as compared to the non-Type D personality and non-depression group at 1 month . At 3 months, with a mean of 5.96, medication adherence was significantly lower in the Type D personality group compared to the non-Type D personality and non-depression group .We used repeated measures ANOVA for mean comparisons of medication adherence among the 4 groups, presented in The relationships between low medication adherence and combined Type D personality and depression are shown in At 1 month, the depression-only group’s medication non-adherence rate was 2.639 times higher  1.170–5.953) than that of the reference group, which was composed of individuals with non-Type D personality and no depression. After adjustment, the medication non-adherence rate was 2.605 times higher (95% CI 1.083–6.266) than that of the reference group.At 3 months, the Type D personality group had a low medication adherence rate that was 4.564 times higher  than that of the reference group.Medication adherence after PCI is a crucial factor to prevent major adverse outcomes such as rehospitalization and mortality ,3,4. RecWith respect to rates of medication adherence, the MMAS-8 cut-off to identify medication non-adherence was 6 ,29,30. IMost importantly, in the present study, medication non-adherence at 1 month was significantly affected only by depression and at 3 months only by Type D personality. After adjusted variables consistent with those of previous studies, such as males, low educational levels, limited exercise, and low perceived good health status, we measured the OR of medication non-adherence. For 1 month after discharge following PCI in this study, patients with depression were 2.6 times more likely to fail to take the medication than the normal group, and at 3 months, 5.1 times higher than the Type D personality group. These results were consistent with previous medication adherence studies about Type D personality and depression. In short, Type D personality alone was a significant predictor of poor medication adherence at 3 or 6 months after discharge ,43. MediAccording to previous studies, many patients might develop depression during or after the diagnosis and treatment of diseases of the heart as a vital organ associated with life . Those wThis present study is meaningful in that it is the first cohort study to examine the combined impact of Type D personality and depression on medication non-adherence during a 3-month follow-up period among PCI patients. However, there was no statistical significance in the relationship between medication non-adherence and combined Type D personality and depression in our study. According to previous studies on cardiovascular disease, Type D personality is highly associated with depression  and TypeIn the present study, we chose a minimum of one- and three-month follow-up periods to identify medication adherence for two reasons: first, a one-month follow-up for medication adherence is a meaningful point in preventing short-term readmissions  after PCI treatment ; second,α of MMAS-8 was 0.58 and indicated little higher than a minimally accepted level as low as 0.50. As known, the MMAS-8 had reported various value of Cronbach’s α from 0.54 to 0.83 in previous studies [This study had some limitations. First, it was based on a small convenience sample, with small numbers of patients with CAD in each group. In future studies, it is necessary to increase the number of samples in conjunction with numerous hospitals in order to obtain many subjects with CAD. Second, the fact that measurements of depression, Type D personality, and medication adherence were self-reported is a big limitation because the findings might underestimate or overestimate the actual rates. Third, we measured Type D personality and depression at baseline with 4 groups. As the prevalence of patients with Type D personality or depression could change at various time points after discharge, regular screening for Type D personality and depression is needed to identify their association with medication non-adherence. Fourth, in this study Cronbach’s  studies ,54,55,56 studies .Our main findings showed that individual Type D personality and depression were risk factors for medication non-adherence in patients who underwent PCI. Therefore, screening for Type D personality and depression at baseline may be useful strategies to improve medication adherence in patients who undergo PCI. Furthermore, nurses should consider integrative person-centered approaches for patients with CAD with a focus on identifying meaningful subgroups by Type D personality and depression.


Aims:Lactobacillus strains have protective effects against heavy metals while relieving oxidative stress and modulating the immune response. Mechanisms that ameliorate heavy metal toxicity and the relationship between probiotics and gut barrier protection in the process of heavy metal pathogenesis was poorly understood.Methods and Results: In this study, Lactobacillus brevis 23017 , a selected probiotics strain with strong mercury binding capacities, was applied to evaluate the efficiency against mercury toxicity in a mouse model. Histopathological results along with HE stains show that L. brevis 23017 protects the integrity of the small intestinal villus, which slows weight loss in response to Hg exposure. The qRT-PCR results demonstrate that L. brevis 23017 maintains a normal mucosal barrier via modulation of tight junction proteins. Importantly, the present study demonstrates that L. brevis 23017 effectively ameliorates injury of the small intestine by reducing intestinal inflammation and alleviating oxidative stress in animal models. Moreover, L. brevis 23017 blocks oxidative stress and inflammation through MAPK and NF-κB pathways, as shown by western blot.Conclusions: Together, these results reveal that L. brevis 23017 may have applications in the prevention and treatment of oral Hg exposure with fermented functional foods by protecting gut health in daily life. Mercury (Hg) is one of the most toxic elements, existing in a variety of sources in the environment, including fish and plants  was purchased from Sigma-Aldrich . Mercury solutions with varying concentrations of HgCl2 were prepared in distilled water. Five groups of 4-week-old female BALB/c mice  were used in the experiments, with each group containing 10 mice. Animal procedures for 5 groups are shown in Table L. brevis 23017 was suspended in MRS medium, and MRS alone served as the control group. The control group (n = 10 per group) was orally dosed with 200 μL MRS medium during the experimental period. (2) The Hg-5d group was dosed with 200 μL MRS medium for the first 5 days, and on the sixth day this group was given oral solutions of Hg at a final concentration of 10 mg/kg (1/5 LD50). (3) The LAB+ Hg-5d group was dosed with L. brevis 23017 (109 CFU/200 μL) for the first 5 days, and on the sixth day this group was given oral solutions of Hg (10 mg/kg). (4) The Hg-12d group was dosed with 200 μL MRS media for the first 5 days, this group was given oral solutions of Hg at a final concentration of 10 mg/kg on the 6th day, and this group continued to be dosed with MRS media for the last 6 days. (5) The LAB+ Hg-12d group was dosed with L. brevis 23017 (109 CFU/200 μL) for the first 5 days, was given oral solutions of Hg at a final concentration of 10 mg/kg on the 6th day, and this group continued to be dosed with L. brevis 23017 (109 CFU/200 μL) for the last 6 days  was obtained from the Institutional Committee of Northeast Agricultural University for animal experiments. Mercury chloride . In brief, pieces of tissue were dried for 4 h at 60°C followed by the addition of 65% HNOIntestinal tissues were collected from standardized areas of the duodenum, jejunum, ileum and colon within 10 to 15 min post mortem. Intestinal and other major organ tissue samples were fixed and stained according to routine histological methods  for 10 min. Supernatants were collected and stored at −80°C for analysis. Intestinal antioxidant activity was determined using a GSH assay kit, SOD assay, CAT assay kit and MDA assay kit, all of which were purchased from Nanjing Jiancheng Bioengineering Institute. Activity of SOD was determined by the epinephrine method  was used to calculate the fold change compared to β-actin gene controls.We employed real-time qRT-PCR to determine the levels of cytokine gene products in intestinal samples using a CFX96TM Real-Time PCR Detection System (Bio-Rad). Total RNA was extracted from intestinal tissues using total RNA extraction kits (iNtRON) according to the manufacturer's instructions. Purified RNA was then converted to cDNA using EasyScript First-Strand cDNA Synthesis SuperMix . The resulting cDNAs were used for real-time PCR with SYBR® qRT-PCR reagent kit , and the specific primers used are listed in Table Intestinal segments were washed with cold PBS and then lysed with RIPA Lysis and Extraction Buffer . Protein concentrations were determined by BCA protein assay kit . One hundred microgram of protein extracts were loaded and electrophoresed on 12% SDS polyacrylamide gels and transferred to PVDF membranes . The following primary antibodies were utilized: anti-P38, anti-p-P38, anti-NF-κB, anti-p-NF-κBand anti-β-actin . The secondary antibody used for detection was conjugated to horseradish peroxidase. Enhanced chemiluminescence (ECL) was used to detect the conjugated horseradish peroxidase. The relative expression of specific bands was analyzed and quantified using ImageJ Software .P < 0.05 was considered statistically significant.All data shown in figures and tables are expressed as the mean ± SEM. One-way ANOVA (SPSS 15.0) was used for data comparisons between multiple groups. In each case, Lactobacillus in the LAB+Hg-5d group decreased more slowly than did the Hg-5d group. The body weights of the LAB+Hg-12d and Hg-12d groups also decreased in response to mercury exposure, but the rate of decrease was not as steep on days 8 and 9 compared with other days. Furthermore, we observed the changes of body weights in the LAB+Hg-12d and Hg-12d groups exhibited a point of inflection on the tenth day of exposure to mercury chloride, with the body weight of mice in the LAB+Hg-12d group increasing on the eleventh day.Changes in body weight were observed among all five groups of mice, as shown in Figure Microscopic examination revealed obvious differences among the five groups of mice with respect to their small intestine samples taken from different intestinal segments. As indicated in Figure p < 0.001). Fecal Hg concentrations of the LAB+Hg-12d group were ~36.21 mg/kg, which were lower than the Hg-12d group (46.28 mg/kg) in the feces. But there was no significant difference between these two groups. In this model of acute mercury poisoning, most of the mercury was discharged in the feces, but the discharged Hg was decreased in the 12-day groups. The feces results show that the Hg concentrations of LAB+Hg-12d and Hg-12d groups were lower than the LAB+Hg-5d and Hg-5d groups. A similar trend was observed for Hg concentrations in the muscle, but Hg concentrations were all significantly lower than in the feces. Most of the Hg was discharged in the feces, that is the reason the muscle absorbed lower Hg concentrations compared with feces. Furthermore, the Hg concentrations of the LAB+Hg-12d group (11.32 mg/kg) in muscle was also lower than the LAB+Hg-5d group (18.13 mg/kg). The Hg-5d group (28.41 mg/kg) also exhibited higher Hg concentration than did the Hg-12d group (21.65 mg/kg). Hg levels of the LAB+Hg-5d and LAB+Hg-12d groups in muscle were significantly higher than in the Hg-5d and Hg-12d groups. Both the LAB+Hg-5d group and LAB+Hg-12d group were significantly lower than the exposure groups(p < 0.05) in terms of Hg levels. Hg concentrations in the muscle highlight the importance of the LAB treatment in mitigating Hg accumulation in the body.Analysis of Hg concentrations in tissue samples and feces revealed dose-dependent increases in Hg levels. As expected, Hg was absent in fecal samples from the control group Figure . The Hg p < 0.05). Compared to Hg-5d, LAB+Hg-5d significantly decreased activities of CAT and MDA, while increasing activities of SOD and GSH, a measure of oxidative stress, in the duodenum and colon (p < 0.05). Preventative assays illustrated that LAB increases CAT and SOD activities in the LAB+Hg-12d group compared to the Hg-12d group in the duodenum and colon (p < 0.05). LAB administration significantly decreased MDA levels in the LAB+Hg-12d group compared to the Hg-12d group in both duodenum and colon. Taken together, these data show that LAB effectively increases SOD and GSH activities and decreases CAT and MDA levels, indicators of oxidative stress. Duodenal SOD results demonstrate that the duodenum plays a more significant role in mediating oxidative stress than does the colon, as duodenal SOD activity was higher than colonic. GSH results suggest that the effects of mercury toxicity are primarily mediated by the duodenum. Oxidative damage in response to mercury toxicity was gradually reduced after the duodenum in the subsequent jejunum, ileum and colon.Activities of MDA, CAT, SOD and levels of GSH were used to evaluate antioxidant levels and are shown in Table p < 0.01) in the duodenum, indicating that LAB preserves duodenal function in response to Hg exposure. ZO-1 expression was also significantly greater in the LAB+Hg-12d group than in the Hg-12d group (p < 0.05). Occludin expression was significantly increased in the duodenum in the LAB+Hg-5d and LAB+Hg-12d groups compared with the Hg-5d and Hg-12d groups, and the occludin levels of Hg-5d and Hg-12d were lower than the control group (p < 0.05) in the duodenum, respectively. However, occludin expression was not significantly different in the colon between the different experimental groups. The mRNA expression of occludin showed that Hg did not affect intestinal integrity in the colon. The results of ZO-1 and occludin indicated that Hg had a different effect on intestinal integrity both in the duodenum and colon. The duodenum and colon have different biological functions.Hg exposure significantly increased the expression of inflammatory cytokines, such as TNF-α, and IL-6, in both the duodenum Figure  and coloNF-κB and their phosphorylated forms were examined by western blot. As expected, Hg and LAB treatment significantly activated both P38 and NF-κB proteins, indicating that both MAPKs and NF-κB protein activation are induced during the acute stage of colonic lesion formation (Figure NF-κB protein activation in the duodenum lesion (Figure NF-κB and proteins were significantly reduced in the duodenum and colon. In the duodenum, the P38, p-P38 and p-NF-κB proteins were inactivated in the LAB groups compared to the Hg groups. In colon, there were significant differences in phosphorylated P38 and NF-κB proteins between Hg and LAB+Hg groups, wherein all protein levels were higher than in the control group, distinct from the duodenum. Hg and LAB treatment did not activate these proteins in the duodenum, whereas 5- and 12-day treatment with LAB were both effective in reducing expression of P38 and p-P38 in the colon. Compared to Hg-12d groups, treatment of LAB was only effective at reducing NF-κB and p-NF-κB expression in the LAB+Hg-12d groups in the colon. There were also significant reductions of P38 and phosphorylated proteins in the LAB+Hg-12d group compared to the Hg-12d group in the colon. In addition, LAB effectively reduced both P38 and p-P38 expression of lesions in the colon induced by Hg exposure. In the NF-κB pathway, exposure of Hg plays a role in chronic injury and activation in the colon, and LAB exerted a long-term blockade of injury in the LAB+Hg-12d group. These results show that LAB primarily inhibits phosphorylation of P38 and NF-κB in the duodenum, which was not the targeted intestinal segment. Exposure of Hg was mostly targeting colonic intestinal segments.To determine the potential role of MAPKs in inflammation observed after LAB and Hg exposure, P38, n Figure . In contn Figure . After pL. brevis 23017, to inhibit Hg absorption in the intestines of mice, and the protective mechanism is partly due to intestinal excretion by the Hg-binding ability of the strain. Hg was absorbed by L. brevis 23017, leading to decreased Hg absorption in muscle and gut. This study was focused on evaluating whether probiotics can protect the gut barrier with other routes on the first targeting tissue of small intestine in addition to Hg absorption with L. brevis 23017.Our previous study confirmed the ability of a probiotic, L. brevis 23017 in the intestines, and an intact gut barrier is crucial to limiting absorption. This study demonstrates the protective effects of L. brevis 23017 in response to Hg exposure, which causes significant damage to the gut barrier, such as damaging enterocytes, increasing intestinal permeability, damaging tight junctions, and inducing oxidative stress and inflammatory responses (Figure Lactobacilli maintains a normal mucosal barrier via modulation of TJ proteins (Dicksved et al., Lactobacillus effectively inhibits weight loss in response to Hg exposure. Histopathological examination illustrated that Lactobacillus attenuates Hg toxicity compared to the control group with a decrease in mucus thickness, supporting a mechanism of increased gut permeability. Both LAB+Hg-5d and LAB+Hg-12d groups maintained relative integrity in the small intestinal villus of the colon, and the intestinal villus in the LAB+Hg-12d group was much longer than in the LAB+Hg-5d group. Hg exposure significantly decreased expression of occludin and ZO-1 in Hg-5d and Hg-12d groups compared to control groups. Furthermore, various studies of heavy metals have demonstrated that they inhibit the growth of diverse microorganisms (Khan et al., Lactobacillus protects the gut barrier against Hg toxicity and is important for inhibition of intestinal Hg absorption, also resulting in protection from the inflammatory response and maintenance of intestinal integrity.There have been many studies on the toxic effects of Hg on different organs, such as the liver, kidneys, muscle, and reproductive system, and toxicity-induced oxidative stress is well documented (Hussain et al., s Figure . PreviouL. brevis 23017 promotes an anti-inflammatory phenotype. L. brevis 23017 administration directly protects the gut from oxidative stress and inflammatory response, while still significantly inhibiting generation of oxidative stress and pro-inflammatory cytokines, reducing MDA levels and increasing activities of SOD, GSH, and CAT. Oxidative stress also aggravates the inflammatory process (Zhai et al., Lactobacillus plantarum inhibits absorption of the heavy metal cadmium by preserving the intestinal barrier (Zhai et al., L. brevis 23017 directly protects against Hg-induced oxidative stress and inflammation.There were many studies demonstrating that Hg exposure induces oxidative stress and inflammatory response in different organs (Lund et al., L. brevis 23017 ameliorates inflammatory bowel disease and that repair of endothelial cells is one of the factors of injury (Feighery et al., Lactobacillus plantarum suppress pro-inflammatory cytokine production via inhibition of NF-kappa B and p38 MAPK expression in murine macrophages (Chon et al., L. brevis 23017 decreases the levels of TNF-α and IL-6, indicating that L. brevis 23017 relieves inflammatory responses during Hg exposure in the early phase treatment. Even though there was no significant difference between the LAB+Hg-5d group and LAB+Hg-12d group in terms of IL-6 and TNF-α expression in the duodenum and colon, the IL-6 and TNF-α expression of the LAB+Hg-12d group was lower than the LAB+Hg-5d group. In addition, the increase of IL-10 expression in the LAB+Hg-12d group was significantly higher than the Hg-12d group in the colon. These results showed that the lactobacillus has the anti-inflammation response for a long period. Lactobacillus curvatus WiKim38 induces IL-10 production in dendritic cells and alleviates DSS-induced colitis in mice (Jo et al., Lactobacillus fermentum ZYL0401 could also attenuates lipopolysaccharide-Induced hepatic TNF-α expression and liver injury via an IL-10 mechanism (Jin et al., Accumulating evidence supports the theory that L. brevis 23017 on NF-κB in response to Hg exposure. These findings demonstrate that the NF-κB pathway may be involved in the pro-inflammatory effects of Hg exposure in the small intestine. However, increased IL-6 and TNF-α levels induced by Hg were not suppressed by L. brevis 23017, suggesting that a unique pathway may exist for Hg to promote the release of IL-6 and TNF-α from the small intestine. Tormos et al., report that p38 MAPK is a sensor of oxidative stress that controls liver regeneration (Tormos et al., L. brevis 23017 blocked the p38 pathway (Hou et al., L. brevis 23017 inhibits Hg exposure-induced oxidative stress and triggers MAPK activation in murine gut cells. Importantly, L. brevis 23017 reduced the levels of pro-inflammatory cytokines and chemokines induced by Hg exposure via down-regulating NF-κB and MAPK activation (Shimazu et al., Additionally, expression of TNF-α, IL-4 and IL-6 were inhibited by NF-κB pathways in different intestinal segments. This finding was supported by our Hg activation of p38 MAPK and NF-κB pathways in the 5- and 12-day groups in the colon, but not the duodenum, which were blocked by L. brevis 23017 oral administration for 5 days and 12 days. Both p38 MAPK and NF-κB pathways were downregulated in response to Hg exposure with concomitant oral administration of L. brevis 23017 for 5 days and 12 days in both the colon and duodenum. Oral L. brevis 23017 blocked the NF-κB pathway much more significantly in the colon compared to the duodenum. NF-κB can be activated by different pathways, including p38 MAPK (Ghosh et al., L. brevis 23017 regulates NF-κB and MAPK pathway activation, we directly measured the effect of L. brevis 23017 on phosphorylation of p38 MAPK. Our results showed that Hg exposure promoted activation both of P38 MAPK, NF-κB and phosphorylation in the colon but not the duodenum. This trend was the same for P38 MAPK and NF-κB pathways. Phosphorylation of p38 MAPK also stimulated phosphorylation of NF-κB. This is the first report that Hg exposure activates p38 MAPK and NF-κB pathways in different intestinal segments. L. brevis 23017 primarily inhibits phosphorylation of mediators such as P38 and NF-κB in the duodenum and colon. In particular, Hg exposure is a cumulative toxicity, and L. brevis 23017 was superior in relieving both P38 and NF-κB pathway activation in the colon.Our results show that Hg selectively activates p38 MAPK and Most organic mercurial compounds are readily absorbed through lungs and GI tract, and some are readily absorbed through the skin. The intestinal epithelium stem cells, which locate in the intestinal mucosa crypts, have the capacity to self-renew for 2–3 days. The intestinal epithelium cells were not the targeting tissue of mercurial compounds. However, chronic effects of mercury were with the thyroid disruption, liver damage, oxidative stress and lipid metabolism disorder on Bufo gargarizans larvae (Shi et al., L. brevis 23017 effectively prevents Hg-induced injury by promoting Hg adsorption in the small intestine by reducing intestinal epithelial cell cytotoxicity, maintaining the integrity of TJ proteins, modulating inflammation and alleviating oxidative stress in an animal model. Most importantly, oral Hg administration only activates P38 and NF-κB pathways in the colon, with differential results in the intestinal segments, and L. brevis 23017 blocks oxidative stress and inflammation through both MAPK and NF-κB pathways. These results provide further insight into the mechanisms of inflammation and oxidative stress, offering a potential target for the prevention and treatment of fermented functional foods against oral Hg exposure to protect gut health in daily life.In conclusion, the present study demonstrates that XJ and JG designed the research. XH and DL analyzed the data. LZ, SG, and SX performed the research. XJ and JG wrote the paper. XH and HC contributed new reagents and analytic tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Long jumpers with below the knee amputation (BKA) have achieved remarkable performances, yet the underlying biomechanics resulting in these jump distances are unknown. We measured three-dimensional motion and used multi-segment modelling to quantify and compare the centre of mass (COM) and joint kinematics of three long jumpers with BKA and seven non-amputee long jumpers during the take-off step of the long jump. Despite having the same jump distances, athletes with BKA, who used their affected leg for the take-off step, had lower sagittal plane hip and knee joint range of motion and positioned their affected leg more laterally relative to the COM compared to non-amputee athletes. Athletes with BKA had a longer compression phase and greater downward movement of their COM, suggesting that their affected leg (lever) was less rigid compared to the biological leg of non-amputees. Thus, athletes with BKA used a different kinematic mechanism to redirect horizontal to vertical velocity compared to non-amputee athletes. The specific movement patterns of athletes with BKA during the take-off step were constrained by the mechanical properties of the prosthesis. These results provide a basis for coaches and athletes to develop training protocols that improve performance and inform the design of future prostheses. Thesen = 8) showed the same basic long jump technique as male non-amputee long jumpers [n = 5, 6.04 ± 0.66 m) compared to those who used their unaffected leg  as their take-off leg [et al. [A previous study reported that male athletes with BKA .et al. [In order to identify differences in foot position, the antero-posterior and medio-lateral distances from the COM to the centre of pressure (COP) of the take-off foot were determined. Additionally, lengths of the whole leg and lower leg were calculated as the linear distance from the COP to the hip joint centre or the linear distance from the COP to the knee joint centre, respectively . All joiet al. .2.4.Due to the small sample size, a non-parametric Wilcoxon ranked-sum test was used to identify differences between the two groups and the margin of error was set to 10% . We assu3.p = 1.000). Athletes with BKA reached MKF in their take-off leg later during the take-off step compared to the non-amputee athletes .The average long jump distance for athletes with BKA (7.26 ± 0.77 m) was not different compared to non-amputee athletes (7.27 ± 0.45 m) (3.1.p = 1.000) nor the distance of the COM relative to the COP at TO (p = 0.517) (p = 0.067). At MKF, both groups had their COM posterior relative to their COP, but athletes with BKA had their COM approximately 13 cm closer to their COP compared to non-amputees (p = 0.017).COM displacement in the antero-posterior (jumping) direction was not different between groups during the take-off step—neither with regard to total displacement (= 0.517)  and 3. Ap = 0.033). Non-amputees had a lateral (relative to their take-off foot) COM displacement (2.6 cm), while athletes with BKA did not have relevant medio-lateral movement during the take-off step .In athletes with BKA, the vertical COM position was below their vertical COM position at TD for a longer relative duration of ground contact compared with non-amputee athletes , but total vertical COM displacement was 18.7% lower (p = 0.017) compared to non-amputee athletes , lost 46.0% less horizontal velocity during stance (p = 0.017) and their horizontal velocity at TO was not different (p = 0.667) compared to non-amputee athletes . Medio-lateral COM velocity was close to zero at TD for both groups. During ground contact, medio-lateral COM velocity was near constant for athletes with BKA, but increased by 0.35 m s−1 in the lateral direction for non-amputee athletes and was different at TO between groups (p = 0.033).Athletes with BKA had 7.5% slower horizontal velocity at TD   and in lower leg length  of the take-off leg were not different in athletes with BKA compared to non-amputee athletes (ctively) .p = 0.017). During the extension phase of the take-off step (MKF–TO), lengthening of the whole leg  and lengthening of the lower leg  were not different between both groups .During the compression phase of the take-off step (TD–MKF), shortening of the whole leg  and shortening of the lower leg  were 159.8% and 194.3% greater, respectively, in athletes with BKA compared to non-amputee athletes (both: 3.2.p = 0.017) (p = 0.017)  . The pasp = 0.117). However, at the instant of MKF, hip flexion was 72.6% lower in athletes with BKA compared to non-amputee athletes (p = 0.017) (p = 0.033) and 40.3% lower at MKF (p = 0.017) but not different at TO (p = 0.517) for athletes with BKA compared to non-amputee long jumpers.Non-amputee athletes had discontinuous hip extension with a flexion angle at TD of 24.8 ± 4.8° and peak hip flexion of 34.4 ± 5.1° at approximately 25% of ground contact during the take-off step. Their hip extension angle at TO was 25.7 ± 3.4°. The sagittal plane hip angle of athletes with BKA displayed a near continuous extension and was not different at TD or TO compared to non-amputee athletes (both = 0.017) . In bothp = 0.183, p = 0.517) and hip  (p = 0.067).Frontal and transverse plane prosthetic ankle angles of athletes with BKA were near constant throughout ground contact of the take-off step, whereas the ankle joint of non-amputee athletes displayed frontal and transverse plane ROMs of 20.6 ± 6.9° and 10.6 ± 3.7°, respectively (= 0.267) . At MKF,4.The purpose of the current study was to quantify three-dimensional long jump take-off step COM and joint kinematics of athletes with BKA and compare those to non-amputee athletes. Long jumpers with BKA, who use their affected leg as their take-off leg, have different COM and joint kinematics throughout the stance phase of the take-off step compared to non-amputee athletes.4.1.During the take-off step, non-amputee long jumpers use a mechanical mechanism called pivoting, where they rotate their COM about their foot to redirect a portion of the horizontal velocity into vertical velocity ,27. Howeet al. [As shown in our previous analysis of the same jumps, ground contact times of the take-off step were not different between non-amputee athletes 122 ± 9 ms) and athletes with BKA 124 ± 14 ms) who used their affected leg as their take-off leg [2 ± 9 ms et al.  define t ms who uet al. ,19. Evenet al. ,19.et al. [et al. [Good long jump performance of non-amputee athletes was related to their ability to tolerate high-impact forces  and to iet al. , but veret al. ,20. Base [et al. , an imme [et al. ,19. Tota [et al. . Thus, l [et al.  argue th [et al. , p. 273 Based on COM kinematics during the take-off step, the determinants of long jump performance are different for non-amputees and athletes with BKA. Further research is needed to identify performance limiting factors for the long jump for athletes with BKA . Knowledge about performance limiting factors may help coaches and biomechanical staff to adapt and enhance the performance analysis for long jumpers with BKA.4.2.Medio-lateral COM position relative to the take-off foot was different throughout the stance phase between groups. The COM position was about 5 cm medial to the COP during most of the take-off step for athletes with BKA, whereas the non-amputee athletes had their COM 0.6 cm medial to the COP at TD and 1.4 cm lateral to the COP at TO. The results from non-amputees are in line with previous studies, which found that the COM was directly above the ankle at TD and then deviated laterally until TO ,20. MediDuring ground contact of the take-off step, non-amputee athletes had increasing lateral velocity, which resulted in a distinct 2.6 cm lateral COM displacement at TO . This la−1 in the lateral direction, shown by the non-amputee athletes  in athletes with BKA  compared to non-amputee athletes   . Apparen4.4.Frontal plane hip angles for non-amputee athletes are in accordance with values reported in previous studies ,20; the To our knowledge, this is the first study to report transverse plane joint angles during the long jump take-off step. Transverse plane hip and knee angles of non-amputee long jumpers are similar to those reported for sprinting . One stu4.5.A limited number of long jumpers with BKA participated in this study; however, these include three out of the four best long jumpers who participated in the 2016 Paralympic Games. Due to the high inter-athlete performance differences in Paralympic long jump, a greater sample size might increase variability and the risk of overlooking movement patterns unique for long jumpers with BKA competing at the highest performance level. Although there are advantages of optoelectrical systems , marker movement on the skin in high-impact movements might influence joint angle calculations. The individual alignment and type of prosthesis used by athletes with BKA probably affect take-off step kinematics. However, all of the RSP models currently used by elite athletes provide the same spring-like function; thus, there are not likely differences in movement strategies between athletes due to different RSP models. In the present analysis, all athletes with BKA used the same type of RSP  with their individual alignment. Future research should investigate the effects of different RSP configurations that optimize fit and performance.5.Long jumpers with BKA positioned their prosthesis more laterally compared to the foot placement beneath the COM used by non-amputee athletes. This strategy may avoid GRF placement on the edge of the prosthesis and, furthermore, may explain the absence of any relevant medio-lateral GRF application  and mediIn our previous paper , we poinIn sum, long jumpers with BKA who use their affected leg as their take-off leg cannot adopt the take-off technique elicited by non-amputee long jumpers and vice versa. Thus, our results can be used to enhance athletic performance of long jumpers with BKA and the design of future prostheses. Additionally, they may inform decision makers during the revision of Olympic and Paralympic regulations.


Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability. MicroRNAs are short (~19–24 nucleotides), single-stranded non-coding RNAs acting as posttranscriptional regulators of gene expression. Cell free microRNAs have been shown to be remarkably stable in blood and other body fluids, as they are released into the extracellular space included in micro vesicles or exosomes or bound to high-density lipoproteins or to the Argonaute2 protein complex , diabeteThis study systematically investigates the impact of selected preanalytical and analytical variables, on the measured plasma levels of miR-92a, miR-126 and miR-16. MiR-92a and miR-126 were selected as they have been suggested as circulating biomarkers in cardiovascular diseases , miR-16 All samples were venous whole blood samples collected into K2-EDTA containing tubes using a 21 gauge needle  after a minimum of venous stasis and discarding of the first 3 mL of blood. The blood samples were centrifuged using a Rotina 420R centrifuge , and all plasma samples were transferred to cryo-tubes and stored at -80°C within 2 hours from blood sampling.Standard plasma (as defined below) and PPP samples from 50 patients with intermittent claudication were collected during June and July 2014 at Lillebaelt Hospital, Kolding, Denmark . All patA total of 30 EDTA anticoagulated whole blood tubes of 5 mL each were obtained from one healthy volunteer in a single venipuncture. The 30 tubes were labeled with consecutive numbers in the order of draw. From each collection tube PPP was prepared by either dual centrifugation (tubes with odd numbers) or prolonged single centrifugation (tubes with even numbers). The dual centrifugation was performed as described above, except that only 0.5 mL was left behind when transferring the plasma. In the prolonged single centrifugation protocol samples were centrifuged at 3,000 g for 30 min , and 1 mL of plasma was left on top of the buffy coat when transferring the PPP to cryo-tubes for storage.All experiments were performed with 3 endogenous microRNAs  and the exogenous cel-miR-39.A schematic overview of the experiments is provided in 1) Dual vs prolonged single centrifugationTo compare the two centrifugation protocols for the preparation of PPP, we performed RT-qPCR (single assays) with purified microRNA from each of 30 PPPs obtained from a healthy volunteer. As described above, 15 of the samples were prepared by dual centrifugation and 15 samples by prolonged single centrifugation. The procedures for microRNA purification and RT-qPCR are outlined below.2) PPP vs standard plasmaCorrelation analysis was used to compare measurements obtained with RT-qPCR (single assays) using patient PPP and standard plasma, respectively.3) Single TaqMan-assays vs TLDACorrelation between measurements obtained by single TaqMan-assays and TLDA was assessed for miR-92a and miR-16. Analysis was performed using the 50 patient PPP samples, and following the procedures described below.4) Comparison of qPCR and ddPCRFirst, the two methods for microRNA quantification were compared with respect to precision and repeatability. A microRNA-pool was prepared from the 30 microRNA samples used above to investigate the centrifugation protocols. In 15 independent analyses on different days, the microRNA-pool was reverse transcribed in doublets, and each of the transcribed cDNAs were assessed, also in doublets, by qPCR (single assays) and ddPCR, respectively. Second, to assess the correlation between measurements obtained by the two methods, microRNA levels in the 50 patient PPP samples were quantified. The procedures for microRNA purification, cDNA synthesis, qPCR and ddPCR are outlined below.−12 M) . MicroRNA was eluted with 30 μL of RNAse-free water, and stored at −80°C.MicroRNA was purified from 300 μL of PPP or standard plasma using Nucleospin®miRNA Plasma  and according to manufacturer’s protocol. After thawing, all archived standard plasmas were re-centrifuged at 3,000 g for 15 min (room temperature) before the microRNA purification, as we have previously shown, that this will minimize the contamination with microRNA from residual platelets in the plasma . To achicDNA synthesis was performed using the TaqMan®MicroRNA Reverse Transcription Kit  and a RT-primer pool containing microRNA-specific stem-loop primers for miR-92a, miR-126, miR-16 and cel-miR-39 . The reaction was performed with 2 µL of purified microRNA in a total volume of 15 μL, and the mixture was incubated at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min as recommended by the manufacturer.-ΔCt (ΔCt = Cttarget-miR−Ctcel-miR-39). Furthermore, Ct-values for miR-92a and miR-126 were also normalized using the endogenous miR-16 or the mean of cel-miR-39 and miR-16. A detailed protocol is available at protocols.io (https://doi.org/10.17504/protocols.io.q9edz3e).Each qPCR contained 1.3 μL transcribed cDNA, 1 μL 20X TaqMan MicroRNA Assay and 10 μL 2X TaqMan Universal PCR Master Mix (Applied Biosystems) in a total volume of 20.3 μL. Each sample was processed in doublets in 40 cycles of 95°C for 15 sec and 60°C for 60 sec using the ABI Prism 7900HT. The mean Ct-values were technically normalized using the exogenous cel-miR-39, and the expression level calculated as 22O. Each ddPCR contained 1.1 μL diluted cDNA, 10 μL 2X ddPCR Supermix for Probes  and 1.0 μL 20X TaqMan MicroRNA Assay (Applied Biosystems) in a total volume of 20 μL. After droplet-generation using the AutoDG (BioRad) the reaction was incubated in 44 cycles of 95°C for 15 sec and 60°C for 60 sec. All samples were run in duplicates, and ddPCR analysis was performed with QX100 Droplet Reader and QuantaSoft Software (BioRad). The mean of the duplicate measurements were normalized to cel-miR-39 or miR-16 by calculating the relative concentration of the target and the reference microRNA. Furthermore, normalization was also performed using the geometric mean of cel-miR-39 and miR-16 as reference. A detailed protocol is available at protocols.io (https://doi.org/10.17504/protocols.io.q8edzte).cDNA synthesis was performed as described above, and the transcribed cDNA was diluted 1:10 with molecular grade HcDNA synthesis was performed using the same kits as described for RT-qPCR (single assays), only the RT-primer pool did not contain the primer for miR-126 but included primers for other microRNAs not used in this study. The reaction was performed using 3 μL of purified microRNA in a total volume of 10 μL, and the mixture was incubated in 40 cycles of 16°C for 2 min, 42°C for 1 min and 50°C for 1 sec, followed by 1 cycle of 85°C for 5 min.As recommended by the manufacturer, a pre-amplification step was performed using 5 μL of the transcribed cDNA in a total reaction volume of 25 μL containing TaqMan®PreAmp Master Mix and Custom TaqMan MIR PreAmp Pool (Applied Biosystems). The pre-amplification mixture was incubated in 14 cycles of 95°C for 15 sec and 60°C in 4 min.https://doi.org/10.17504/protocols.io.q62dzge).The final quantification was performed using Custom TaqMan®Array MicroRNA Cards (TLDA) from Applied Biosystems. A total of 1.2 μL of the pre-amplification products were mixed with TaqMan®Universal Master Mix II, no UNG and loaded on the array. The samples were assessed in triplets in 40 cycles of 97°C for 30 sec and 59.7°C in 60 sec using the ABI Prism 7900HT, and the mean Ct-values were normalized as described for the single assay RT-qPCR. A detailed protocol is available at protocols.io . Normalization was performed as described above for qPCR and ddPCR using either the spiked-in cel-miR-39, the endogenous miR-16 or a combination of both. The Student t-test was used to compare the microRNA levels obtained with the two centrifugation protocols. A sample size calculation was performed in order to ascertain the number of samples needed to detect a difference of 10% in the mean microRNA level between the two centrifugation methods. All samples were drawn from the same volunteer in a single venipuncture, thus we considered the samples to be paired. Using a standard deviation of 20% of the mean microRNA level  and a correlation coefficient of 0.85 we needed 12 samples in each centrifugation protocol (significance level 0.05 and power 0.8).The Spearman’s rank correlation coefficient (rho) was used to compare the results obtained by RT-qPCR (single assays) to results obtained by RT-qPCR (TLDA) and RT-ddPCR, respectively. Furthermore, results obtained by RT-qPCR using microRNA purified from PPP and standard plasma were compared. P-values below 0.05 were considered as statistical significant.Precision (inter-assay variability) and repeatability (intra-assay variability) of microRNA measurements were estimated with coefficient of variation [CV = (SD/mean)*100] of the duplicate measurements from 15 independent analyses.When microRNA levels were normalized to cel-miR-39, no significant differences in levels of miR-92a (p = 0.35), miR-126 (p = 0.26) or miR-16 (p = 0.25) between plasmas obtained by the two centrifugation protocols were found. When normalized to the endogenous miR-16 or to the mean of cel-miR-39 and miR-16, we also found no significant differences in levels of miR-92a (p≥0.52), whereas levels of miR-126 were significantly higher in PPP when obtained by dual centrifugation compared to prolonged single centrifugation (P≤0.02),Coefficients of variation were found to be high with both centrifugation strategies and higher when microRNA levels were normalized to cel-miR-39 (10.9–21.1%) as compared to normalization using miR-16 (9.5–14.2%) or a combination of cel-miR-39 and miR-16 (9.2–13.3%), When plotting the microRNA levels of the 30 PPPs, we see no clear pattern to indicate that the measured microRNA levels are influenced by the drawing order, When using cel-miR-39 as a mean of normalization, we found no correlation between measurements obtained with PPP and standard plasma as starting material (rho≤0.28 and p≥0.05). When microRNA levels were normalized to miR-16, we found intermediate correlation between measurements obtained with the two starting materials, rho = 0.42 (p = 0.0024) for miR-92a and rho = 0.48 (p = 0.0004) for miR-126. An intermediate correlation was also observed, when miR-126 levels were normalized to a combination of cel-miR-39 and miR-16 , whereas with this normalization strategy we found no correlation for miR-92a , . When levels of miR-16 were normalized to cel-miR-39, correlation between the two methods were found to be intermediate ,  to be significantly lower with qPCR as compared to ddPCR, except for miR-92a when normalized to miR-16 or to the mean of cel-miR-39 and miR-16. Results are outlined in Correlation of measurements obtained by qPCR and ddPCR were found to be very good when microRNA levels were normalized to cel-miR-39 . When microRNA levels were normalized to miR-16 or to the mean of cel-miR-39 and miR-16 the correlation between the two methods was still very good for miR-126 , but only moderate for miR-92a , total−CVRT-qPCR)/CVtotal, where CVtotal is the mean of the CVs estimated for the dual and prolonged single centrifugations .We compared microRNA-levels in PPP obtained by dual and prolonged single centrifugation. When microRNA levels were technically normalized to cel-miR-39 we found no significant differences between results obtained using the two centrifugation protocols, and we also found no differences in miR-92a levels when normalized to the endogenous miR-16 or to a combination of the two reference microRNAs. In contrast, when normalized to miR-16 or to the two reference microRNA in combination, we found miR-126 levels to be significantly higher in PPP obtained by dual centrifugation. Nevertheless, in PPP the level of miR-16 was found to be 256 fold  higher than the level of miR-126, which makes miR-16 less suitable as a mean of normalization of miR-126 levels. By way of comparison, there were 8–16 fold  differences in levels of miR-16 and miR-92a, and only 4 fold  differences in levels of cel-miR-39 and miR-126. Coefficient of variation was found to be high, and largely independent of the centrifugation protocol used. Furthermore, though one might speculate that due to platelet activation or hemolysis, microRNA levels in the blood collection tubes may increase over the time course of the blood sampling procedure, when using a minimal of stasis, we found no significant impact on measured microRNA-levels of the order of draw. To our knowledge this is the first study to provide evidence for the possibility to use a single centrifugation step for preparation of PPP, and that the order of blood draw has no significant impact on the results of microRNA analyses.We have previously shown that the plasma levels of a number of microRNAs are higher in standard plasma compared to PPP . In the MicroRNAs have been found to be promising biomarkers for a wide variety of diseases, but in many cases the results are never validated by others, which in part may be due to the differences in the microRNA-platform used . We compCampomenosi et. al. used ddPCR (EvaGreen) and qPCR (SYBR Green) to perform absolute quantification of 3 microRNAs in serum, and found very good correlation between results obtained by the two techniques  . In the In the study by Campomenosi et. al., aliquots of cDNA-samples were used to compare the inter-assay precision of microRNA measurements obtained with ddPCR and qPCR. They found in microRNA-126 analysis the mean CV to be 7.94% and 7.39%, respectively . Other sWe found that together, the centrifugation and purification steps in our microRNA analysis accounted for 64–73% of the intra-assay variation when using cel-miR-39 for data normalization, 39–66% when data were normalized to miR-16 and 55–61% when a combination of the two reference microRNAs were used. In line with this, McDonald et. al. found RNA-extraction to account for 77–92% of the variation in their microRNA-analyses .The strengths of our study are that the same set of samples was used to compare the different platforms, that the same patient cohort was used to compare PPP to standard plasma, and that all analyses were performed by the same scientist. Furthermore, the intra- and inter-assay variations were estimated using results from 15 independent duplicate analyses each performed with purified microRNA as starting material. The relatively low number of microRNA examined may be a limitation, and the choice of references for normalization may influence the results obtained, though the same reference miRNAs were used in all the methods compared.In conclusion, our results highlight the importance of a comprehensive description of the preanalytical and analytical factors used, when publishing results from microRNA studies, in order to allow for others to perform the exact same experiments in validation studies.Furthermore, when conclusions are made regarding observed differences in microRNA-levels between healthy and diseased subjects or between groups of patients, the high intra- and inter-assay variability of the analyses must be taken in to account. If microRNAs are to become routinely used biomarkers in a clinical setting, their quantification must be reproducible, and differences in plasma levels must be of a magnitude large enough to overcome the impact of preanalytical and analytical variability.S1 FigParts A-C and J shows the correlation between microRNA measurements obtained by single TaqMan assays (single qPCR) and TaqMan Low Density Array (TLDA). Parts D-I and K shows the correlation between microRNA measurements in PPP and standard plasma (single TaqMan assays). In parts A, D, G, J and K the target miRNAs are normalized to cel-miR-39, in parts B, E and H miRNA-16 was used for normalization, and in parts C, F and I data are normalized to the mean of cel-miR-39 and miR-16.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.Cyclic Adenosine 3′,5′-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2.  Coxiella burnetii, the etiological agent of the Q-fever, is an obligate intracellular bacterium. This microorganism has a biphasic life cycle in which the small cell variant (SCV) and the large cell variant (LCV) alternate. The LCV form is generated in the lysosomal-like large replicative compartment  of infected cells, where the acidic microenvironment favors its replication . In . In Coxim (T4SS)  6]. The. TheCoxiynthesis . We haveynthesis . Furtherynthesis  9]..CoxiellaThe autophagy pathway is a highly conserved, physiological degradation process in eukaryotic cells. During autophagy, small portions of cytoplasm or damaged organelles are sequestered into double-membrane vesicles named autophagosomes. These vesicles then fuse with degradatives organelles which supply the hydrolytic enzymes for breaking down and eventual recycling of the sequestered material. The Microtubule-associated protein light chain 3 (LC3) has been shown to be an autophagosomal marker in mammals. There are two forms of LC3 called LC3-I and LC3-II, which are produced post-translationally in various cell types. LC3-I is a cytosolic protein, whereas LC3-II is membrane-bound and specifically associates with autophagosome membranes.Staphylococcus aureus and Legionella pneumophila, exploit the autophagy pathway for their own benefit in order to survive and replicate in the host cells.  [12 [1211] . Th. Th18]. S. aureus . Th. Th20]. . aureus . That st. aureus .C. burnetii co-opt to generate its replicative vacuole. The results obtained in this work offer a deeper insight into the molecular components of the host cell that are involved in the regulatory mechanism of the development of C. burnetii replicative vacuole.In this work, we aimed at studying the effectors EPAC and Rap2b as key regulators of CRV development. We have demonstrated that the cAMP modulated protein EPAC was recruited to the CRV. In addition by analyzing the EPAC downstream effector Rap2b, we determined that the latter factor, but not its inactive mutant Rap2b ΔAAX, is also recruited to the CRV from early times p.i. More importantly, we demonstrated that over-expression of Rap2b wt protein, but not Rap2b ΔAAX, significantly impaired the development of the large CRV. Interestingly, we have shown that the over-expression of Rap2b wt reduced both, the homotypic and the heterotypic fusion capacity of the CRV, and also, decreased the intracellular levels of the v-SNARE Vamp7. These results suggest that the over-expression of the active form of Rap2b affects molecular components of the fusion machinery that Coxiella antiserum and mCherry-Coxiella burnetii were generously provided by Dr. Robert Heinzen . Coxiella-GFP (Tn1832) and the DotA mutant GFP (Tn292) were kindly provided by Dr. Matteo Bonazzi . Texas Red-tagged dextran (3 kDa) was purchased from Molecular Probes. PEGFP-Rap2b wt and pEGFP-Rap2b ΔAAX plasmids were kindly provided by Dr. MauroTorti . Plasmids for EPAC-GFP, Δ(1–148)-EPAC-GFP, Δ(72–148)-EPAC-GFP were kindly provided by Dr. Xiaodong Cheng .D-MEM and alpha-MEM were obtained from Gibco Laboratories ; fetal bovine serum (FBS) was obtained from GIBCO BRL/Life Technologies . The anti-Rap2b antibody and Rap2b siRNA were purchased from Santa Cruz Biotechnology . Rabbit anti-2 atmosphere until 80% confluence was reached.Vero cells , HeLa cells  and Chinese hamster ovary cells (CHO)  were grown in 24-well plates in either D-MEM or α-MEM, supplemented with 15% FBS, and the antibiotics streptomycin (50μg/ml) and penicillin (50 μg/ml), at 37ºC in a 5% COCHO cells were transfected with the plasmids (1 μg/μl) using the Lipofectamine 2000 reagent  as previously described in . TransfeC. burnetii clone 4, phase II, Nine Mile strain was performed as previously described , . Th. ThS. auC. burnetii (homotypic fusion) or vesicles from the endocytic, autophagic and secretory pathways (heterotypic fusion). As demonstrated before, an inhibition of fusion causes a decrease in the size of the CRVs and therefore increases the number of smaller vesicles containing C. burnetii 35].[35].34][.[35].34] pathway . The mulC. burnetii was not able to fuse with lysosomes or with other CRV in the absence of Vamp7 . We . We 38].of Vamp7 . Since CCoxiella vacuole and that this effect might be at least in part, by modulating the levels of Vamp7. This is a very intriguing observation. However, further studies would be necessary to determine the critical molecular mechanisms involved in this modulation.In conclusion, in this report we have demonstrated that the cAMP effectors EPAC and Rap2b are key regulators of CRV development. We have proved that the small GTPase Rap2b has a critical role in the development of CRV by altering the fusion events of the S1 Table(PDF)Click here for additional data file.


Of particular interest in this regard is a rapid escape, or startle, reflex possessed by zebrafish larvae; this reflex, the C-start, is mediated by a relatively simple neuronal circuit and exhibits habituation, a non-associative form of learning. Here we demonstrate a rapid form of habituation of the C-start to touch that resembles the previously reported rapid habituation induced by auditory or vibrational stimuli. We also show that touch-induced habituation exhibits input specificity. This work sets the stage for  Interesin vivo –13, maketh nerve, which forms highly specialized synapses, known as club synapses, on the distal lateral dendrites of the M-cell [Brief auditory pulses elicit a short-latency startle response, the C-start, in zebrafish; this response results from selective activation of one of a pair of large hindbrain command neurons, the Mauthner cells (M-cells) . By conte M-cell . These ce M-cell . Stimulae M-cell , which fe M-cell . Althouge M-cell –21, habi4, 0.33 mM CaCl2, 0.17 mM KCl, 10−5% methylene blue, pH 7.2), and incubated at 28.5°C. All experiments were conducted with the Tüpfel long fin (TL) wild-type strain of zebrafish obtained from the University of California, Los Angeles (UCLA) core facility. A Casio Exilim ExFH25  was used for all behavioral experiments; images were recorded at 240 frames/s. During the experiments, fish were placed on a light box  to allow sufficient contrast to observe the escape responses.Standard breeding protocols were used in accordance with UCLA Animal Research Care (ARC) requirements . Zebrafish eggs were collected, placed into E3 solution  larval zebrafish were embedded in 3.5% low melting point agarose in a cell culture dish. After the agarose had solidified, E3 (or drug-containing E3) was added to the dish. The tail of the fish was freed from the agarose to permit it to move, and a portion of agarose above the head was removed to permit electrical or mechanosensory stimulation of the head . In expeThe same stimuli used to test the responsiveness of the larval zebrafish were used to induce habituation. After allowing 30 min for the fish to acclimate to the experimental situation, the threshold intensity for eliciting the C-start was determined ; 15 min later habituation training commenced. The training comprised 120 electrical/mechanosensory stimuli delivered at 1 Hz. The response of the fish—whether or not a C-start was elicited—to the first training stimulus was used as the pretest response; posttests were given 10 s–5 min after training, depending on the training protocol. Control fish received a single pretest and a single posttest stimulus delivered at the equivalent experimental times as the pretest and posttest for the trained fish.Two stimulating electrodes were placed on either the same side or opposite sides of the head of a larva. For same-side stimulation, electrodes were placed approximately 1.5 mm apart in order to stimulate different receptive fields of trigeminal sensory neurons. For both same-side and opposite-side placement, one of the two electrodes was used to deliver habituating stimuli, while the other electrode was used only for posttest stimulation (There was no pretest stimulation in these experiments). One of the electrodes (training or control) delivered a posttest stimulus 10 s after the end of training; a second posttest stimulus was delivered 5 s later via the other electrode. Which electrode was used for the 10-s posttest and which for the later posttest was counterbalanced between the training and control electrodes.Larval zebrafish were treated through bath application with Strychnine hemisulfate salt  or control vehicle solutions 18–24 h prior to testing unless otherwise indicated. Fish were maintained in the same solution throughout an experiment.t-tests or ANOVAs. Paired t-tests were used for within subject comparisons, whereas unpaired t-tests were used to compare different groups. Repeated measures ANOVAs were used to assess the significance of differences between groups that were tested at three different times. Significant interactions were further probed using a one-way ANOVA. Post-hoc analyses of significant ANOVAs were performed using Tukey HSD tests.Comparisons were performed using either Shock-10 s, 0.17 ± 0.167; TrainedShock-30 s, 0.29 ± 0.184; TrainedShock-1 min, 0.29 ± 0.184; TrainedShock-5 min, 1.00 ± 0.000) and fish that only received the test stimulus  revealed that the 10-s, 30-s, and 1 min posttest groups were each significantly different (p < 0.05 for each comparison) from the TrainedShock-5 min group and the Test AloneShock group. These data indicate that this form of habituation persists for 1 min, but not 5 min. Thus, the habituation induced was short-lasting. This form of habituation resembles the rapid form previously reported for AV stimulation [Two forms of behavioral plasticity of the C-start have been previously described in zebrafish larvae: auditory/vibrational (AV) stimulation-elicited habituation and dishabituation , 19–21. WP) received only the pretest and posttest. Groups of trained fish received the single posttest at various times after training . As observed in the experiments using electrical shocks, head-directed water pulses elicited robust, but short-lasting, habituation. An ANOVA comparing the response rate of fish given habituation training with water pulses  to fish that only received the test stimulus  revealed a significant difference at the 10-s posttest (p < 0.05) compared to all groups other than the Trained WP-30 s group. These data indicate that this form of habituation lasts for 10–30 s, but not longer.We also examined habituation of the startle response to water pulses , 22 Fig. The taiOPP) or only the posttest (Test AloneOPP). Posttests were delivered to the training and test alone sides of the heads in a counterbalanced manner to avoid order effects. Following habituation training, the TrainedOPP response (0.125 ± 0.125) was significantly (p < 0.05) reduced compared to the Test AloneOPP response (0.750 ± 0.160)  . Thus, tSS), whereas the other delivered only the posttest stimulation (Test AloneSS). We observed that the posttest response to stimulation with the TrainedSS electrode (0.150 ± 0.080) was significantly less than that of the stimulation with the Test AloneSS electrode . This result indicates that untrained neural circuit maintains significant responsiveness and that, therefore, the habituation induced by the electrical stimulation was partly, if not fully, input-specific.The habituation produced by unilateral skin shocks might have been due to pathway-specific homosynaptic depression or feedforward inhibition of the sensory-neuron-to-command-neuron circuit ; alternaTC group) exhibited a significantly altered escape response within 60 min compared to a vehicle-treated control group (E3TC) (T) or untrained (StrychUT) larvae   and that in the group given only the test stimuli after the strychnine treatment  was not significant (p = 0.15) (Marsden and Granato  reportedp (E3TC) . Nonethe) larvae . Eightee < 0.05) . The dif = 0.15) . These rWe have found that application of shocks or water pulses to the head of zebrafish larvae elicits a startle response from them whose short latency is consistent with mediation by an M-cell or M-cell analog , 20, 22.We also determined that habituation to weak head shocks is at least partly input-specific. Specifically, we showed that the habituation of the C-start to training on one side of the larva’s head did not generalize to the opposite side, as indicated by the lack of significant habituation to the opposite-side test stimulus . On the In summary, the present study indicates that touch-induced habituation of the C-start reflex in zebrafish larvae exhibits input-specificity and generalization, two hallmarks of habituation , 33. MorS1 Fign = 7) after habitation training with AV stimulation (Trained AV) to their mean response rate to the posttest electrical stimulus (Untrained StimElectrode). The response rate to the AV stimulus was significantly less than that to the electrical stimulus . (b) Comparison of the responsiveness of restrained larvae (n = 8) following habitation training with electrical shocks to the skin (Trained StimElectrode) to their responsiveness to the posttest AV stimulus (Untrained AV). The responsiveness of the larvae following the two types of experimental manipulation differed significantly .(a) Comparison of the mean response rate of restrained larvae ((TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


In the present study, we fabricated a dual-mode cardiac troponin I (cTnI) biosensor comprised of multi-functional DNA (MF-DNA) on Au nanocrystal (AuNC) using an electrochemical method (EC) and a localized surface plasmon resonance (LSPR) method. To construct a cTnI bioprobe, a DNA 3 way-junction (3WJ) was prepared to introduce multi-functionality. Each DNA 3WJ arm was modified to possess a recognition region (Troponin I detection aptamer), an EC-LSPR signal generation region (methylene blue: MB), and an anchoring region (Thiol group), respectively. After an annealing step, the multi-functional DNA 3WJ was assembled, and its configuration was confirmed by Native-TBM PAGE for subsequent use in biosensor construction. cTnI was also expressed and purified for use in biosensor experiments. To construct an EC-LSPR dual-mode biosensor, AuNCs were prepared on an indium-tin-oxide (ITO) substrate using an electrodeposition method. The prepared multi-functional (MF)-DNA was then immobilized onto AuNCs by covalent bonding. Field emission scanning electron microscope (FE-SEM) and atomic force microscopy (AFM) were used to analyze the surface morphology. LSPR and electrochemical impedance spectroscopy (EIS) experiments were performed to confirm the binding between the target and the bioprobe. The results indicated that cTnI could be effectively detected in the buffer solution and in diluted-human serum. Based on the results of these experiments, the loss on drying (LOD) was determined to be 1.0 pM in HEPES solution and 1.0 pM in 10% diluted human serum. Additionally, the selectivity assay was successfully tested using a number of different proteins. Taken together, the results of our study indicate that the proposed dual-mode biosensor is applicable for use in field-ready cTnI diagnosis systems for emergency situations. As the obesity rate and the overall age of the population are both increasing worldwide, concerns of higher incidences of cardiovascular diseases (CVDs) are gradually growing . CVDs arSeveral biomarkers are capable of detecting AMI, and these include cardiac troponin , myoglobin (MB), creatine kinase-MB, and MB isozymes ,7. AmongCurrently, several types of cTnI detection methods have been developed to detect cTnI sensitively and selectively ,11. The To solve these problems, for the first time, we fabricated a cTnI dual detection biosensor composed of a multi-functional DNA 3 way-junction (3WJ)-based bioprobe immobilized onto an Au nanocluster (AuNC)-modified ITO substrate using EC-LSPR methods. To harness the required multi-functionality, nucleic acid possessed various advantages such as ease of tailoring and the ability to regulate their length, compared to antibodies. However, the multi-functionality of conventional double strand-based nucleic acid is limited due to the number of arms. To simultaneously perform three functions, DNA 3WJ is suitable for target binding, signal reporting, and anchoring to substrate ,21,22. T4·3H2O), Triton-X solution, ammonium sulfate, potassium citrate tribasic monohydrate, and sodium hydroxide were purchased from Sigma-Aldrich . The three fragments of DNA 3WJ were provided by Bioneer . The sequence of the cTnI aptamer was utilized according to the method provided by the Ban group [To synthesize AuNC on the ITO substrate, Hydrogen tetrachloroaurate(III) trihydrate . The PCR was performed with the conditions previously described by Jo et al. [6-tag at the N-terminal. An E. coli strain (DH5a) was used as the host for subcloning. Standard subcloning techniques were performed in this work. The forward primer was designed to have a BamHI restriction enzyme site, and the reverse primer was designed to contain a HindIII restriction enzyme site. The PCR product was isolated using a DNA purification kit , and digested with two restriction enzymes for BamHI and HindIII . The digested DNA fragments were ligated into a pET-28a vector  that was previously digested with BamHI and HindIII using a ligation kit . To obtain the cTnI protein, the pET28a vector encoding cTnI gene was transformed into E. coli BL21 (DE3) [The cTnI gene fragments were amplified from HEK293 cDNA by polymerase chain reaction (PCR) using purchased oligonucleotide primers with o et al. . The gen21 (DE3) ,26. The 4, 0.5 mM ammonium sulfate, and 2 mM potassium citrate tribasic monohydrate. The potential was applied at −1.3 V (vs. Ag/AgCl) for a fixed time (120 s), and the deposition was performed at 25 °C. After the deposition, the ITO electrode was removed from the electrochemical cell, rinsed with DIW, and air-dried for further use [AuNC was electrochemically synthesized using a potentiostat . For the three-electrode setup, an ITO-coated glass substrate  was used as a working electrode, a platinum wire was used as the counter electrode, and Ag/AgCl served as the reference electrode. ITO-coated glass substrates were cleaned by sonication for 30 min using 2% Triton X-100 solution, deionized water (DIW), and ethanol. AuNC was electrochemically deposited onto ITO substrates (30 mm × 10 mm) using 5 mM HAuCl2, 100 mM NaCl) by heating at 80 °C for 5 min, followed by slowly cooling down to 4 °C at a rate of 2 °C/min on a T100™ Thermal Cycler . The assembly of prepared DNA bioprobes was confirmed by our previous study [To assemble multi-functional DNA structure, DNA 3WJ fragments were modified with a functional group. Tro6/3WJa (5′-CGC ATG CCA AAC GTT GCC TCA TAG TTC CCT CCC CGT GTC CTT GCC ATG TGT ATG TGG G-3′) was introduced. For the generation of simultaneous plasmonic signal and electrochemical signal, MB-tagged DNA 3WJb was added . Finally, thiol-modified DNA 3WJc was prepared to allow for anchoring of the AuNC directly . All above DNA fragments were all chemically synthesized by Bioneer . The AIapt/MB/SH-3WJ was finally assembled by annealing three corresponding DNA strands at an equal molar ratio in TMS buffer , the roughness max (Rmax), the roughness average (Ra) and the vertical distance.The AuNC-modified ITO substrates were analyzed by FE-SEM , and the biofilm formation was investigated by AFM . The bare ITO substrate, AuNC-modified ITO, DNA 3WJ self-assembled onto AuNC-modified ITO substrate, and the HA protein/DNA 3WJ/AuNC-modified ITO substrates were all characterized for comparison. Prior to scanning the sample, all the conditions such as the set point current, integrated gain, and proportional gain were optimized to the force between the tip and the substrate surface . The AFMTo optimize the immobilization condition of the Tro6/MB/SH-DNA 3WJ, the LSPR method was carried out. The cTnI protein binding to the Tro6/MB/SH-DNA 3WJ was analyzed by the peak shift of LSPR in the UV–Vis spectrum resulting from the changes in the local refractive index induced by the target-aptamer reaction. The local refractive index changes by target-aptamer interaction at a given wavelength took place because of the extension of light absorption of the biofilm on the AuNC-modified ITO electrode. All absorption spectra were observed by monitoring UV–Vis spectral changes in the transmission mode using a JASCO V-530 UV-spectrometer .et). The electrode response can be expressed as (Ri − Ro)/Ro, where Ro depicted the electron transfer resistance of the fully modified electrode obtained from the dimethylamine buffer prior to aptamer immobilization and Ri represented the electron transfer resistance of the fully modified electrode obtained from the specific concentration of the aptamer-target in the same buffer. All electrochemical measurements were performed using a Versastat3 potentiostat  [To perform cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments, a customized-three-electrode system setup was utilized. The DNA 3WJ/AuNC-ITO electrode served as the working electrode. Pt electrodes and Ag/AgCl electrodes were mounted on the top of the fabricated electrode used as a counter electrode and reference electrode, respectively. CV and EIS were performed in 0.5 M of dimethylamine. The potential for CV was cycled from −0.9 V to 0.9 V at a 0.05 V/s scan rate. The EIS analysis was set to measure the electron transfer resistance in the frequency ranging from 1 Hz to 100 kHz under a potential of 0.2 V. The potential was decided as a redox potential that was determined by the initial CV analysis . The colOR, USA) .a value was 1.048 ± 0.287 nm, the RMS roughness (Rq) was 2.335 ± 0.714 nm, the Rmax was 3.539 ± 1.222 nm, and the vertical distance (VD) between the surface and the top of the ITO was 12.545 ± 3.882 nm. After AuNC formation on the ITO substrate by the electrodeposition process, the grain size was increased to 60 to 90 nm compared to ITO substrate, which is similar to that observed in the FE-SEM image of AuNC-modified ITO substrate  was 12.579 ± 3.514 nm, the Rmax was 24.628 ± 7.245 nm, and the vertical distance (VD) between ITO substrate and the top of AuNC was 38.256 ± 7.984 nm. When the multi-functional DNA 3WJ was immobilized , RbulkLSPR measurements were performed on the prepared DNA 3WJ/AuNC-modified electrode after that addition of cTnI. To analyze the cTnI detection performance in the context of loss on drying (LOD), dynamic range, and selectivity, LSPR experiments were conducted on eight samples. Additionally, the fabricated LSPR cTnI biosensor was tested with cTnI in diluted human serum to determine clinical suitability. After the washing step, the cTnI was reacted with the DNA 3WJ/AuNC-modified ITO electrode for 2 h at RT. The obtained LSPR bands corresponded to the serially diluted cTnI concentrations . The various concentration changes in cTnI dissolved in PBS buffer were observed by the absorbance changes as refractive index (RI) changes on the AuNC surface. Increment of absorbance intensity values and redshifts in the wavelengths of LSPR bands were monitored when the cTnI concentration was increased a. These Rct measured for the biosensor prior to cTnI detection, as to denoted as R0  as shown in (i (~20%), and the observed increases in Ri linearly varied with the cTnI concentration range of 1.0 pM to 10 nM. For our system that used a dimethylamine buffer, the LOD value was determined as 497 fM in buffer through error bar and slopes of  in dimethylamine buffer. The typical responses of the biosensor equipped with the cTnI measured by EIS are shown in a. The Rcshown in b. The hilopes of b. This iented in c, and thto 10 nM d. The prto 10 nM d, indicai − R0/R0). This result can be explained by the possibility that only the cTnI specifically reacted with MF-DNA on the AuNC electrode to drastically increase the resistance. Additionally, the detection performance comparison of the fabricated EIS-LSPR dual biosensor and other previously reported biosensors is summarized in (rized in  [29,30,33, MoS2 and Bi2Se3, not only AuNC [To rapidly treat AMI patients, the construction of a portable AMI detection system is essential. For the first time, the present study proposed a highly sensitive, label-free cTnI detection biosensor composed of multi-functional DNA on AuNC-modified ITO substrate that functions through a EC-LSPR dual detection method. To incorporate three functionalities into one DNA structure, DNA 3WJ serves as a suitable cTnI biosensor. A cTnI aptamer, a MB group, and a thiol group were sequentially attached to the end of the DNA 3WJ fragment. In particular, the MB acted to simultaneously generate the EC signal and enhance the LSPR signal. The assembled DNA 3WJ also retained multi-functionality. The AuNC-modified ITO substrate was prepared using the electrodeposition method, and the surface morphology was confirmed by FE-SEM. AFM and surface roughness analyses were also used to examine the immobilization process of fabricated biosensor. The LSPR and EIS experiments were performed to confirm the cTnI binding to the DNA 3WJ/AuNC-modified electrode. The LODs for cTnI were validated in both PBS buffer and diluted human serum, respectively. The LOD value was determined to 110 fM in buffer and 840 fM in diluted serum through LSPR experiments, respectively. Also, the LOD value was determined to 497 fM in buffer and 829 fM in diluted serum through EIS experiments, respectively. Based on these results, the proposed EC-LSPR dual detection biosensor composed of multi-functional DNA and AuNC exhibits simplicity, label-free detection, high sensitivity, and selectivity for the clinical assessment of AMI disease. Moreover, the proposed dual detection system can be tuned to sensitivity by introducing the plasmonic nanomaterials such as MoOnly AuNC ,33,34. A